The impact of polyether chain length on the iron clearing efficiency and physiochemical properties of desferrithiocin analogues

Article abstract of DOI:10.1021/jm9018146

(S)-2-(2,4-Dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylic acid (2) was abandoned in clinical trials as an iron chelator for the treatment of iron overload disease because of its nephrotoxicity. However, subsequent investigations revealed that replacing the 4′-(HO) of 2 with a 3,6,9-trioxadecyloxy group, ligand 4, increased iron clearing efficiency (ICE) and ameliorated the renal toxicity of 2. This compelled a closer look at additional polyether analogues, the subject of this work. The 3,6,9,12-tetraoxatridecyloxy analogue of 4, chelator 5, an oil, had twice the ICE in rodents of 4, although its ICE in primates was reduced relative to 4. The corresponding 3,6-dioxaheptyloxy analogue of 2, 6 (a crystalline solid), had high ICEs in both the rodent and primate models. It significantly decorporated hepatic, renal, and cardiac iron, with no obvious histopathologies. These findings suggest that polyether chain length has a profound effect on ICE, tissue iron decorporation, and ligand physiochemical properties.

Full text of DOI:10.1021/jm9018146

J. Med. Chem. 2010, 53, 2843–2853 2843
DOI: 10.1021/jm9018146
The Impact of Polyether Chain Length on the Iron Clearing Efficiency and Physiochemical
Properties of Desferrithiocin Analogues
Raymond J. Bergeron,*,† Neelam Bharti,† Jan Wiegand,† James S. McManis,† Shailendra Singh,† and Khalil A. Abboud‡
†
Department of Medicinal Chemistry University of Florida, Gainesville, Florida 32610-0485, and ‡Department of Chemistry,
University of Florida, Gainesville, Florida 32611
Received December 8, 2009
(S)-2-(2,4-Dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylic acid (2) was abandoned in clin-
ical trials as an iron chelator for the treatment of iron overload disease because of its nephrotoxicity.
However, subsequent investigations revealed that replacing the 40-(HO) of 2 with a 3,6,9-trioxadecyloxy
group, ligand 4, increased iron clearing efficiency (ICE) and ameliorated the renal toxicity of 2. This
compelled a closer look at additional polyether analogues, the subject of this work. The 3,6,9,12-
tetraoxatridecyloxy analogue of 4, chelator 5, an oil, had twice the ICE in rodents of 4, although its ICE
in primates was reduced relative to 4. The corresponding 3,6-dioxaheptyloxy analogue of 2, 6 (a crystalline
solid), had high ICEs in both the rodent and primate models. It significantly decorporated hepatic, renal,
and cardiac iron, with no obvious histopathologies. These findings suggest that polyether chain length has a
profound effect on ICE, tissue iron decorporation, and ligand physiochemical properties.
Introduction
failure is still the leading cause of death in thalassemia major
and related forms of transfusional iron overload.18-20
In humans with iron overload disease, the toxicity asso-
ciated with an excess of this metal derives from iron’s inter-
action with reactive oxygen species, for instance, endogenous
hydrogen peroxide (H2O2).1-4 In the presence of Fe(II), H2O2
is reduced to the hydroxyl radical (HO•), a very reactive
species, and HO-, the Fenton reaction. The hydroxyl radical
reacts very quickly with a variety of cellular constituents and
can initiate free radicals and radical-mediated chain processes
that damage DNA and membranes as well as produce car-
cinogens.2,5,6 The Fe(III) liberated can be reduced back to
Fe(II) via a variety of biological reductants (e.g., ascorbate,
glutathione), a problematic cycle.
The iron-mediated damage can be focal, as in reperfusion
damage,7 Parkinson’s,8 and Friedreich’s ataxia,9 or global, as
in transfusional iron overload, e.g., thalassemia,10 sickle cell
disease,10,11 and myelodysplasia,12 with multiple organ in-
volvment. The solution in both scenarios is the same: chelate
and promote the excretion of excess unmanaged iron. The
design, synthesis, and evaluation of ligands for the treatment
of transfusional iron overload diseases represent the focus of
the current study.
While humans have a highly efficient iron management
system in which they absorb and excrete about 1 mg of iron
daily, there is no conduit for the excretion of excess metal.
Transfusion-dependent anemias, like thalassemia, lead to
a build up of iron in the liver, heart, pancreas, and else-
where, resulting in (i) liver disease that may progress to
cirrhosis,13-15 (ii) diabetes related both to iron-induced
decreases in pancreatic β-cell secretion and to increases in
hepatic insulin resistance,16,17 and (iii) heart disease. Cardiac
Treatment with a chelating agent capable of sequestering
iron and permitting its excretion from the body is the only
therapeutic approach available. Some of the iron-chelating
agents that are now in use or that have been clinically
evaluated include desferrioxamine B mesylate (DFOa),21
1,2-dimethyl-3-hydroxy-4-pyridinone (deferiprone, L1),22-25
4-[3,5-bis(2-hydroxyphenyl)-1,2,4-triazol-1-yl]benzoic acid (de-
ferasirox, ICL670A),26-29 and the desferrithiocin, (S)-4,5-dihy-
dro-2-(3-hydroxy-2-pyridinyl)-4-methyl-4-thiazolecarboxylic
acid (DFT, 1, Table 1), analogue, (S)-2-(2,4-dihydroxy-
phenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylic acid [deferi-
trin (2),30 Table 1]. Each of these ligands presents with serious
shortcomings. DFO must be given subcutaneously for pro-
tracted periods of time, e.g., 12 h a day, five days a week, a
serious patient compliance issue.31-33 Deferiprone, while orally
active, simply does not remove enough iron to maintain patients
in a negative iron balance.22-25 Deferasirox did not show
noninferiority to DFO and is associated with numerous side
effects; it has a very narrow therapeutic window.26-29 Finally,
the clinical trial on 2 (Table 1) was abandoned by Genzyme
because of renal toxicity.30 However, deferitrin (2) has been
reengineered, leading to the discovery that replacing the 40-
hydroxyl on the aromatic ring of 2 with a 3,6,9-trioxadecyloxy
polyether group solved the renal toxicity issue;34 iron clearing
efficiency (ICE) was also improved. These findings drive the
current study, which focuses on the impact of polyether chain
length on ligand iron clearing efficiency and toxicity. The
ultimate goal is to identify a chelator that is orally active in an
a Abbreviations: dec, decomposes; DFO, desferrioxamine B mesy-
late; DFT, desferrithiocin [(S)-4,5-dihydro-2-(3-hydroxy-2-pyridinyl)-
4-methyl-4-thiazolecarboxylic acid]; DIEA, N,N-diisopropylethyla-
mine; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide;
ICE, iron-clearing efficiency; log Papp, octanol-water partition coeffi-
cient (lipophilicity); MIC, maximum iron clearance.
*To whom correspondence should be addressed. Phone: (352) 273-
7725. Fax: (352) 392-8406. E-mail: rayb@ufl.edu. Address: Box 100485
JHMHSC, Department of Medicinal Chemistry, University of Florida,
Gainesville, Florida 32610-0485.
r
2010 American Chemical Society
Published on Web 03/16/2010
pubs.acs.org/jmc

2844 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Bergeron et al.
Table 1. Iron-Clearing Efficiency of Desferrithiocin Analogues Administered Orally to Rodents and Primates with the Respective Log Papp Values and
Physiochemical Properties
a In the rodents [n = 3 (6), 4 (3-5, 7), 5 (1), 8 (2)], the drugs were given po at a dose of 150 μmol/kg (1) or 300 μmol/kg (2-7). The drugs were
administered in capsules (6, 7), solubilized in either 40% Cremophor RH-40/water (1), distilled water (4), or were given as their monosodium salts,
prepared by the addition of 1 equiv of NaOH to a suspensionof the free acid in distilled water (2, 3, 5). The efficiency of each compound was calculated by
subtracting the 24 h iron excretion of control animals from the iron excretion of the treated animals. The number was then divided by the theoretical
output; the result is expressed as a percent. The ICE data for ligand 1 is from ref 39. The ICE data for 2-4 are from ref 34. b ICE is based on a 48 h sample
collection period. The relative percentages of the iron excreted in the bile and urine are in brackets. c In the primates [n = 4 (1, 3, 4, 5, 6 in capsules, 7) or 7
(2, 6 as the monosodium salt)], the chelators were given po at a dose of 75 μmol/kg (5-7) or 150 μmol/kg (1-4). The drugs were administered in capsules
(6d, 7), solubilized in either 40% Cremophor RH-40/water (1, 3), distilled water (4), or were given as their monosodium salts, prepared by
the addition of 1 equiv of NaOH to a suspension of the free acid in distilled water (2, 5, 6e). The efficiency was calculated by averaging the iron
output for 4 days before the drug, subtracting these numbers from the 2 day iron clearance after the administration of the drug, and then dividing
by the theoretical output; the result is expressed as a percent. The ICE data for ligand 1 is from refs 40, 41. The ICE data for 2-4 are from refs 42, 43,
and 34, respectively. The relative percentages of the iron excreted in the feces and urine are in brackets. f Performance ratio is defined as the mean
ICEprimates/ICErodents.
g Data are expressed as the log of the fraction in the octanol layer (log Papp); measurements were done in TRIS buffer, pH 7.4,
using a “shake flask” direct method.52 The values for 2 and 3 are from ref 43; the value for 4 is from ref 34. h The mp data for 1-3 are from ref 39, 42,
and 43, respectively.
acceptable dosage form, is very efficient at decorporating iron,
and has a broad therapeutic window. The boundary condition
set by many hematologists is that the chelator should be able to
remove 450 μg/kg/day of the metal.35
chelators shown to be orally active. It performed well in both
the bile duct-cannulated rodent model (ICE, 5.5%)39 and in
the iron-overloaded Cebus apella primate (ICE, 16%).40,41
Unfortunately, 1 was severely nephrotoxic.41 Nevertheless,
its outstanding oral activity spurred a structure-activity
study to identify an orally active and safe DFT analogue.
The first goal was to define the minimal structural platform,
pharmacophore, compatible with iron clearance upon oral
administration.42-44
Results and Discussion
Design Concept. DFT (1) is a natural product iron chela-
tor, a siderophore. It forms a tight 2:1 complex with Fe(III),
has a log β2 of 29.6,36-38 and was one of the first iron

Article
Removal of the pyridine nitrogen of DFT provided (S)-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2845
Ultimately, we discovered that fixing a polyether moiety, a
3,6,9-trioxadecyloxy group, to the 40-position of 2, providing
(S)-4,5-dihydro-2-[2-hydroxy-4-(3,6,9-trioxadecyloxy)phenyl]-
4-methyl-4-thiazolecarboxylic acid (4, Table 1), resulted in a
ligand that retained the ICE properties of 3 but was much less
lipophilic and less toxic than 3.34 This polyether fragment has
been fixed to one of three positions on the aromatic ring, 30-,
40-, or 50-.34,46 The iron-clearing efficiency in rodents and
primates is shown to be very sensitive to which positional
isomer is evaluated.34,46 In rodents, the polyethers had
uniformly higher ICEs than their corresponding parent
ligands. There was also a profound reduction in toxicity,
particularly renal toxicity.34,46,47 In the primate model, the
ICEs for both the 30- and 40- polyethers were similar to the
corresponding phenolic parent, e.g., the 30-(HO) isomer of
deferitrin (2) and 2, respectively.46 However, the ICE of
the 50-polyether substituted ligand decreased relative to its
parent.46 What remained unclear was the quantitative sig-
nificance of the length of the polyether backbone on the
properties of the ligands, the subject of this work.
In the current study, additional polyether analogues of 2
were synthesized (Table 1). Specifically, the 3,6,9-trioxa-
decyloxy substituent at the 40-position of ligand 4 was both
lengthened to provide (S)-4,5-dihydro-2-[2-hydroxy-4-(3,
6,9,12-tetraoxatridecyloxy)phenyl]-4-methyl-4-thiazolecar-
boxylic acid (5) and shortened to provide (S)-4,5-dihydro-2-
[2-hydroxy-4-(3,6-dioxaheptyloxy)phenyl]-4-methyl-4-thia-
zolecarboxylic acid (6). The ethyl ester of 6, ethyl (S)-4,
5-dihydro-2-[2-hydroxy-4-(3,6-dioxaheptyloxy)phenyl]-4-meth-
yl-4-thiazolecarboxylate (7), was also prepared. Three ques-
tions were addressed regarding the structural changes in
ligand 2: (1) the effect on lipophilicity, (2) the effect on the
iron clearing efficiency in the bile duct-cannulated rodent and
primate models, and (3) the effect on the physiochemical
properties ofthe ligand. Wehaveconsistently seen that, within
a given family, ligands with greater lipophilicity are more
efficient iron chelators, but are also more toxic,34,43,45 thus
issues 1 and 2. We have also observed that the polyether acids
for the 30- and 40-3,6,9-trioxadecyloxy analogues are oils, and
in most cases, the salts are hygroscopic. A crystalline solid
ligand would offer greater flexibility in dosage forms.
4,5-dihydro-2-(2-hydroxyphenyl)-4-methyl-4-thiazolecar-
boxylic acid [(S)-DADFT],44 the parent ligand of the desaza
(DA) series. Substitution of the 4-methyl of (S)-DADFT
with a hydrogen led to (S)-4,5-dihydro-2-(2-hydroxyphenyl)-
4-thiazolecarboxylic acid[(S)-DADMDFT],41,44 the platform
for the ensuing DADM systems. In the course of additional
structure-activity relationship (SAR) studies, we were
able to determine that within a given family of ligands, e.g.,
the DADFTs or the DADMDFTs, that the chelator’s log
Papp, lipophilicity, had a profound effect on both ICE and
toxicity.34,43,45 In each family, as the lipophilicity decreases,
i.e., the log Papp becomes more negative, the toxicity also
decreases. The more lipophilic chelators generally had greater
ICE and increased toxicity.34,43,45 It is critical to remain
within families when making these comparisons. For exam-
ple, there is no relationship between the log Papp, ICE, and
toxicity of DFT itself versus the log Papp, ICE, and toxicity of
its analogues. However, in the case of the desaza family of
ligands, for example, when a 40-(CH3O) group was fixed in
place of the 40-(HO) of 2, providing (S)-4,5-dihydro-2-(2-
hydroxy-4-methoxyphenyl)-4-methyl-4-thiazolecarboxylic
acid (3, Table 1), the molecule’s lipophilicity increased, as
did its ICE and toxicity.34,43 This ligand is very lipophilic,
log Papp= -0.70, and a very effective iron chelator when
given orally to rodents34 or primates43 (Table 1). Unfortu-
nately, the ligand was also very nephrotoxic.34 The question
then became how to balance the lipophilicity/toxicity inter-
action while iron-clearing efficiency is maintained.
Scheme 1. Synthesis of (S)-4,5-Dihydro-2-[2-hydroxy-4-(3,6,9,12-
tetraoxatridecyloxy)phenyl]-4-methyl-4-thiazolecarboxylic Acid (5)a
Synthesis. Deferitrin (2) was converted to ethyl (S)-2-(2,4-
dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarbox-
ylate (10)48 in this laboratory. With the carboxylate group
protected as an ester, alkylation of the less sterically hindered
40-hydroxy of 10 in the presence of the 20-hydroxy, an iron
a Reagents and conditions: (a) NaI (2 equiv), acetone, reflux, 18 h,
94%; (b) 9 (1.3 equiv), K2CO3 (1.3 equiv), acetone, reflux, 2 d, 73%;
(c) 50% NaOH (aq) (11 equiv), CH3OH, 94%.
Scheme 2. Synthesis of (S)-4,5-Dihydro-2-[2-hydroxy-4-(3,6-dioxaheptyloxy)phenyl]-4-methyl-4-thiazolecarboxylic Acid (6) and Its
Ethyl (7) and Isopropyl (13) Estersa
a Reagents and conditions: (a) K2CO3 (1.1 equiv), acetone, reflux, 2 d, 73%; (c) 50% NaOH (aq) (13 equiv), CH3OH, 80%; (c) 2-iodopropane
(1.6 equiv), DIEA (1.6 equiv), DMF, 3 d, 85%.

2846 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Bergeron et al.
chelating site, has generated numerous desferrithiocin ana-
logues, including 3-6 (Table 1).34,43
Thus, O-monoalkylation of ethyl ester 10 with 13-iodo-
2,5,8,11-tetraoxatridecane (9) using potassium carbonate in
refluxing acetone generated masked chelator 11 in 73% yield
(Scheme 1). Tetraether iodide 9 was readily accessed in 94%
yield from tosylate 8,49,50 employing sodium iodide (2 equiv)
in refluxing acetone, as alkylating agent 8 possesses similar
chromatographic properties to ester 11. Hydrolysis of the
ester protecting group of 11 in base completed the synthesis
of 3,6,9,12-tetraoxatridecyloxy ligand 5, a homologue of 447
with an additional ethyleneoxy unit in the polyether chain, in
94% yield.
Figure 1. X-ray of (S)-4,5-dihydro-2-[2-hydroxy-4-(3,6-dioxaheptyl-
oxy)phenyl]-4-methyl-4-thiazolecarboxylic acid (6). Structure is
drawn at 50% probability ellipsoids.
The synthesis of the 3,6-dioxaheptyloxy ligand (6), the
analogue of chelator 4 with one less ethyleneoxy unit in
the polyether chain, was prepared using similar strategy
(Scheme 2). 40-O-Alkylation of ethyl ester 10 with 3,6-
dioxaheptyl 4-toluenesulfonate (12)49 generated 7 in 73%
recrystallized yield. Unmasking the carboxylate of 7 under
alkaline conditions furnished the shorter 40-polyether-derived
iron chelator 6 in 80% recrystallized yield. Both ligand 6 and its
ethyl ester 7 are crystalline solids and thus offer clear advan-
tages both in large scale synthesis and in dosage forms over
previously reported polyether-substituted DFTs, which are
oils.34,46,47 Carboxylic acid 6 was esterified using 2-iodopro-
pane and N,N-diisopropylethylamine (DIEA) (1.6 equiv each)
in DMF, providing isopropyl (S)-4,5-dihydro-2-[2-hydroxy-
4-(3,6-dioxaheptyloxy)phenyl]-4-methyl-4-thiazolecarboxylate
(13) in 85% yield as an oil (Scheme 2). This is consistent with
the idea that the structural boundary conditions for ligand
crystallinity are very narrow.
Figure 2. X-ray of ethyl (S)-4,5-dihydro-2-[2-hydroxy-4-(3,6-diox-
aheptyloxy)phenyl]-4-methyl-4-thiazolecarboxylate (7). Structure
is drawn at 50% probability ellipsoids.
focused on minimizing toxicity while optimizing iron clear-
ance are carried out.
Physiochemical Properties. Single crystal X-ray analysis
confirmed that chelator 6 (Figure 1) and its ethyl ester 7
(Figure 2) exist in the (S)-configuration. Both 6 and 7
crystallize in monoclinic lattices, space group P21, with
two molecules in unit cell. Moreover, acid 6 has unit cell
dimensions of a = 5.5157(5) A, b = 8.8988(8) A, and c =
17.3671(16) A with R and γ = 90° and β = 98.322(1)°. The
unit cell dimensions of ester 7 are a = 7.7798(6) A, b =
8.9780(6) A, and c = 14.1119(10) A, also with R and γ = 90°
but β = 106.078(1)°. Unit cell volumes (A3) of 6 and 7 are
843.46(13) and 947.12(12), respectively. In the crystal lattice
of 6, the acidic hydrogen is bonded to O6A of the carboxylate
group, resulting in a neutral molecule (Figure 1). However,
parent ligand 2 (Table 1) with a strongly electron donating 40-
hydroxy is zwitterionic, that is, an iminium ion is observed by
X-ray crystallography.51 Thus, not unexpectedly, deferitrin
(2) (log Papp = -1.05) is more hydrophilic than polyether
chelator 6 (log Papp = -0.89).
Partition Properties. The partition values between octanol
and water (at pH 7.4, Tris buffer) were determined using a
“shake flask” direct method of measuring log Papp values.52
The fraction of drug in the octanol is then expressed as
log Papp. These values varied widely (Table 1), from log
Papp= -1.77 for 1 to log Papp= 3.00 for 7. This represents a
greater than 58000-fold difference in partition. The most
lipophilic chelator, 7, is 11220 times more lipophilic than the
parent 2.
Chelator-Induced Iron Clearance in Non-Iron-Overloaded,
Bile Duct-Cannulated Rodents. In the text below, “iron-
clearing efficiency” (ICE) is used as a measure of the amount
of iron excretion induced by a chelator. The ICE, expressed
as a percent, is calculated as (ligand-induced iron excretion/
theoretical iron excretion) ꢀ 100. To illustrate, the theo-
retical iron excretion after administration of one millimole of
DFO, a hexadentate chelator that forms a 1:1 complex with
Fe(III), is one milli-g-atom of iron. Two millimoles of
desferrithiocin (DFT, 1, Table 1), a tridentate chelator which
forms a 2:1 complex with Fe(III), are required for the
theoretical excretion of one milli-g-atom of iron. In the
rodents, in each instance, the polyether analogues are better
iron clearing agents than their phenolic counterparts, e.g., 2
vs 4, 5, 6, or 7 (Table 1). We have included historical data
(compounds 1-4)34,39,43 for comparative purposes. The ICE
of the 3,6,9-trioxadecyloxy analogue (4) is five times greater
than that of the parent ligand (2), 5.5 ( 1.9% vs 1.1 ( 0.8%
(p < 0.003), respectively.34 The longer ether analogue,
3,6,9,12-tetraoxatridecyloxy analogue (5), is nearly 11 times
as efficient as 2, with an ICE of 12.0 ( 1.5% (p < 0.001). The
shorter ether analogue, the 3,6-dioxaheptoxy ligand (6), and
its corresponding ethyl ester (7), are highly crystalline solids
that were administered to the rats in capsules.56 Both ligands
are approximately 24 times as effective as the parent 2, with
ICE values of 26.7 ( 4.7% (p < 0.001) and 25.9 ( 6.5% (p <
0.001), respectively. The difference in iron clearing proper-
ties between 4 and 5 vs 6 and 7 is likely due to the differences
in lipophilicity as reflected in the log Papp (Table 1). This
observation has remained remarkably consistent throughout
our studies with DFT analogues.34,43,45 The latter two
ligands are more lipophilic, with larger log Papp values.
Animal Models. There are no dependable in vitro assays
for predicting the in vivo efficacy of an iron decorporation
agent.53,54 While tight iron binding is a necessary require-
ment for an effective iron chelator, it is not sufficient.55 Once
having established that a ligand platform, pharmacophore,
binds iron tightly, e.g., desferrithiocin,37,38 SAR studies

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2847
Figure 3. Biliary ferrokinetics of DFT-related chelators 2 and 4-7 given orally to non-iron-overloaded, bile duct-cannulated rats at a dose of
300 μmol/kg. The iron excretion (y-axis) is reported as μg of iron per kg body weight.
The biliary ferrokinetics profiles of the ligands, 2 and 4-7,
are very different (Figure 3) and clearly related to differences
in the polyether backbones. The maximum iron clearance
(MIC) of the parent drug, deferitrin (2), occurs at 3 h, with
iron clearance virtually over at 9 h. The trioxa polyether (4)
also has an MIC at 3 h, with iron excretion extending out to
12 h. The tetraoxa ether analogue 5 has an MIC at 6 h; iron
excretion continues for 24 h. The MIC of the dioxa ether
analogue 6 and its corresponding ester 7 do not occur until
12-15 h, and iron excretion had not returned to baseline
levels even 48 h postdrug. Note that although the biliary
ferrokinetics curve of 6 may appear to be biphasic (Figure 3),
the reason for this unusual line shape is that several animals
had temporarily obstructed bile flow. While the concentra-
tion of iron in the bile remained the same, the bile volume,
and thus overall iron excretion, decreased. Once the obstruc-
tion was resolved, bile volume and overall iron excretion
normalized.
Chelator-Induced Iron Clearance in Iron-Overloaded Pri-
mates. The iron clearance data for the chelators in the
primates are described in Table 1. We have included historical
data (compounds 1-4) for comparative purposes.34,39,40,42,43
Ligand 2 had an ICE of 16.8 ( 7.2%,34 while the ICE of 4 is
25.4 ( 7.4%.34 The ICE of the longer 3,6,9,12-tetraoxa ana-
logue (5) was significantly less, 9.8 ( 1.9% (p < 0.001). The
shorter 3,6-dioxa analogue, 6, had an ICE of 26.3 ( 9.9% when
it was given to the primates in capsules; the ICE was virtually
identical when it was administered by gavage as its sodium salt,
28.7 (12.4% (p > 0.05). The similarity in ICE of 6 between the
encapsulated acid and the sodium salt given by gavage sug-
gest comparable pharmacokinetics. The ester of ligand 6,
compound 7, performed relatively poorly in the primates, with
an ICE of only 8.8 ( 2.2%.
tively (Table 1).46 In the current study, the PR of ligand 5 is
0.8, while that of 6 is 1.0. Previously, the only ligand that
behaved so alike in primates and rodents was the 50-isomer
of 4, which also had a performance ratio of 1.46 However,
on an absolute basis, the ICE for this chelator in primates
(8.1 ( 2.8%) was, in fact, poor. In current study, ligand 6
performed exceptionally well in both rodents and primates
(ICE > 26%), suggesting a higher index of success in
humans. The ester of 6, ligand 7, on the other hand, had
a very low performance ratio (0.3), lower than we have
previously observed.
The profound difference between the ICE of the parent
acid chelator 6 vs that of the ester 7 in rodents and primates is
consistent with two possible explanations: (1) the ester is
poorly absorbed from the gastrointestinal (GI) tract in the
primates, or (2) the primate nonspecific serum esterases
simply may not cleave ester 7 to the active chelator acid 6.
An experiment was performed using rat and monkey plasma
in an attempt to determine if the relatively poor ICE of 7 in
the primates was due to interspecies differences in hydrolysis.
When 7 was solubilized in DMSO and incubated at 37 °C
with rat plasma, all of the ester had been converted to the
active acid 6 within 1-2 h. This was also the case when the
experiment was carried out with plasma from the Cebus
apella monkeys. Thus, there is no difference in the hydrolysis
of 7 between the rats and the primates. Therefore, the poor
ICE of 7 in the monkeys is consistent with the idea that the
ester is absorbed much more effectively from the GI tract of
the rodents than from the GI tract of the primates. Control
experiments were also performed in which saline was used in
place of the rat or monkey plasma. Note that when 7 was
solubilized in DMSO and incubated with saline in place of
the rat or monkey plasma, all of the drug remained in the
form of the ester.
There are some very notable differences between the
current ICE data and previously reported studies.34,43,46
In the past, ligands generally performed significantly better
in the iron-overloaded primates than in the non-iron-
overloaded rodents. For example, we reported that the
Toxicity Profile of (S)-4,5-Dihydro-2-[2-hydroxy-4-(3,6-
dioxaheptyloxy)phenyl]-4-methyl-4-thiazolecarboxylic Acid
(6) and its Ethyl Ester (7). Ten-day toxicity trials have been
carriedoutinratsonbothligands6and 7. The drugs were given
to the animals orally once daily at a dose of 384 μmol/kg/d
(equivalent to 100 mg/kg/d of DFT sodium salt). Additional
performance ratio (PR), defined as the mean ICEprimates
/
ICErodents, of analogues 2-4 are 15.3, 3.7, and 4.6, respec-

2848 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Bergeron et al.
Figure 4. Tissue iron concentration of rats treated with 6 once daily at a dose of 384 μmol/kg/d ꢀ 10 d. The chelator was administered orally in
gelatin capsules (n = 5) or by gavage as its monosodium salt (n = 10). Age-matched rats (n = 12) served as untreated controls.
Figure 5. Tissue iron concentration of rats treated with 7 once daily at a dose of 192 (n = 6) or 384 μmol/kg/d (n = 5) ꢀ 10 d. The chelator was
administered orally in gelatin capsules. Age-matched rats (n = 12) served as untreated controls.
age-matched animals served as untreated controls. The animals
were euthanized on day 11, one day after the last dose of drug.
Extensive tissues were sent out for histopathological examina-
tion. The kidney, liver, pancreas, and heart of test and control
animals were removed and wet-ashed to assess their iron
content.
Because ligand 6 was such an effective iron chelator in
both the rats and the primates, its toxicity profile is most
relevant. The key comment from the pathologist was that
“The tissues from rats in group 1 [test group] cannot be
reliably differentiated histologically from the tissues from
rats in group 2 [control animals].” This was very encourag-
ing, especially in view of how much iron the chelator
removed from the liver and heart in such a short period of
time (discussed below). However, in spite of this outcome, it
is clear that any protracted toxicity trials in rodents will have
to include groups of both iron-loaded and non-iron-loaded
animals, as a 28-day exposure to 6 could reduce the liver iron
stores sufficiently to lead to toxicity.
The scenario with the ethyl ester of 6, compound 7, was
somewhat different. While its ICE was excellent in rodents,
along with an impressive reduction in liver and renal iron
content (discussed below), ester 7 did present with some
renal toxicity. Mild to moderate vacuolar degeneration of
the proximal tubular epithelial cells was found when 7 was
given at a dose of 384 μmol/kg/d ꢀ 10 d. However, when the
dose of 7 was reduced to 192 μmol/kg/d ꢀ 10 d, there were no
drug-related abnormalities.
Tissue Iron Decorporation. As described above, rodents
were given acid 6 or 7 orally at a dose of 384 μmol/kg/d ꢀ10 d.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2849
Ethyl ester 7 was also given at a dose of 192 μmol/kg/d ꢀ
10 d. On day 11, the animals were euthanized and the kidney,
liver, pancreas, and heart were removed. The tissue samples
were wet-ashed, and their iron levels were determined,
Figures 4 and 5. The renal iron content of rodents treated
with 6 was reduced by 7.4% when the drug was administered
in capsules and by 24.8% when it given as its sodium salt
(Figure 4). Although the renal iron content of the latter
animals was significantly less than that of the untreated
controls (p < 0.001), there was not a significant difference
between the capsule or sodium salt groups (p > 0.05). The
reduction in liver iron was profound, >35% in both the
capsule and sodium salt groups (p < 0.001). There was a
significant reduction in pancreatic iron when the drug was
given as its sodium salt (p < 0.05) vs the untreated controls,
but not when it was dosed in capsules (Figure 4). However,
as with the renal iron, there was no significant difference
between the capsule vs sodium salt treatment groups (p >
0.05). Finally, there was a significant decrease in the cardiac
iron of animals treated with acid 6, 6.9% and 9.9% when the
drug was given in capsules and as its sodium salt, respectively
(p < 0.05).
Rats given the ethyl ester 7 in capsules orally at a dose
of 384 μmol/kg/d ꢀ 10 d had a profound reduction in both
renal and hepatic iron vs the untreated controls, 32.1% (p <
0.001) and 59.1% (p < 0.001), respectively (Figure 5). We
have never observed such a dramatic decrease in tissue iron
concentration. Because of the renal toxicity observed with 7
at the 384 μmol/kg/d dosing regimen, we decided to repeat the
10-day toxicity study, this time administering the drug at half
of the dose, 192 μmol/kg/d. A clear dose response was
observed in the reduction in renal and liver iron concentra-
tions (Figure 5). The kidney iron reduction was 32.1% at 384
μmol/kg/d, and 12.6% at 192 μmol/kg/d (p < 0.01). The liver
iron reduction was 59.1% at 384 μmol/kg/d and 27% at 192
μmol/kg/d (p < 0.001). Neither dose was associated with a
reduction in pancreatic or cardiac iron content.
alkylated with either polyetheriodide9 or tosylate 12toafford
11 or 7, respectively. This was followed by hydrolysis of each
ethyl ester in aqueous base, providing 5 (an oil) with a longer
polyether chain (Scheme 1), or ligand 6, possessing a shorter
polyether chain (Scheme 2). Both 6 and its ester 7 are crystal-
line solids. The toxicity profile, efficacy as an iron-clearing
agent, and physiochemical state, a crystalline solid, make
ligand 6 a very attractive clinical candidate. The fact that
the ethyl ester of 6, masked ligand 7, also readily crystallizes is
remarkable (see X-ray structures, Figures 1 and 2). All poly-
ether analogues previously synthesized by this laboratory,
both acids and esters, were oils.34,46,47 In most instances, metal
salts of the former were hygroscopic. Interestingly, even the
isopropyl ester of 6, compound 13, was an oil. Because 6 and 7
are crystalline solids, they were given in capsules56 to both the
rodents and the primates.
In rodents, the ICE of 5 as its sodium salt was nearly 11
times greater than that of the parent (2) and twice as effective
as the trioxa polyether (4). The shorter polyether acid 6 given
in capsules had an ICE that was 24 times greater than 2 and
was nearly five times greater than that of 4 (Table 1). The
ICE of the corresponding ester 7 was virtually identical to
that of 6. The biliary ferrokinetics curves for both 6 and 7
were profoundly different than any of the other ligands
(Figure 3). MIC did not occur until 12-15 h postdrug, and
iron clearance was still ongoing even at 48 h. In contrast,
MIC occurred much earlier with the other ligands, 3 h for 2
and 4, and 6 h for 5. In addition, iron excretion had returned
to baseline levels by 9 h for 2, 12 h for 4, and 24 h for 5
(Figure 3). If the protracted iron clearance properties of
ligand 6 were also observed in humans, thalassemia patients
may only need to be treated two to three times a week. This
would be an improvement over the rigors of the currently
available treatment regimens.
In primates, the ICE of the parent polyether 4 was 2.5
greater than thatof the longer analogue5, while the ICE of the
shorter polyether analogue 6 was within error of that of 4
(Table 1). However, the ICE of the ethyl ester of 6, ligand 7, is
only one-third that of 6 (Table 1). Studies in rat and monkey
plasma suggested no difference in the nonspecific esterase
hydrolysis of 7 between the rats and the primates. The poor
ICE of 7 in the monkeys is, however, consistent with the idea
that the ester is absorbed much more effectively from the GI
tract in rodents than in primates.
The protracted biliary ferrokinetics and outstanding iron
clearing efficiencies of polyether acid 6 and ester 7 noted in the
bile duct-cannulated rats (Figure 3) were reflected in a dra-
matic reduction in the tissue iron levels of rodents treated
orally with the drugs once daily for 10 days (Figures 4 and 5).
Acid 6, given orally in capsules or by gavage as its sodium salt,
significantly reduced both hepatic and cardiac iron (Figure 4)
with no histological abnormalities noted between the treated
and the control groups. Compound 7 administered in capsules
decorporated even more iron from the kidney and liver than 6
but had no impact on pancreatic or cardiac iron burden
(Figure 5). However, this is probably a moot point, as ester
7 presented with unacceptable renal toxicity.
Conclusion
Earlier studies with 2 revealed that methylation of the 40-
hydroxyl resulted in a ligand (3) with better ICE in both the
rodents and the primates (Table 1).43 However, ligand 3 was
unacceptably nephrotoxic34 and was reengineered, adding a 3,6,9-
trioxadecyl group to the 40-(HO) in place of the methyl.34,46,47 This
resulted in a chelator (4) with about the same ICE in rodents and
primates as methylated analogue 3, but virtually absent of any
nephrotoxicity.34 The corresponding 30- and the 50-trioxa ana-
logues also had better ICE properties in rodents than the 40-O-
methyl ether 3. In the primates, the ICE of the 30-trioxa ligand was
similar to that of the 40-trioxa analogue (4), while the 50- was less
effective. These data encouraged an assessment of how altering the
length of the polyether chain would affect a ligand’s ICE,
lipophilicity, and physiochemical properties.
The 3,6,9-trioxadecyloxy substituent at the 40-position of
ligand 434 was both lengthened to a 3,6,9,12-tetraoxatridec-
yloxy group, providing 5, and shortened to a 3,6-dioxahep-
tyloxy moiety, providing 6. In addition, the ethyl (7) and
isopropyl (13) esters of ligand 6 were also generated. The
synthetic methodologies were very simple with high yields, an
advantage when large quantities of drug are required for
preclinical studies.
Compound 11 (Scheme 1), the ethyl ester of chelator 5
(Table 1), was an intermediate in the synthesis of 5. We elected
not to evaluate ester 11 because the parent acid itself did
not perform well in primates. The ester, even if cleaved to the
acid 5 in animals by nonspecific serum esterases, would not
be expected to perform any better than the parent acid itself.
This is underscored when comparing acid 6 (Table 1) with its
In all cases, the ethyl ester of 2, compound 10, served as the
starting material (Schemes 1 and 2). The 40-(HO) of 10 was

2850 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Bergeron et al.
ester 7 (Table 1). This ester does not work as well in primates
as the parent acid. This is also why we elected not to evaluate
ester 13. The synthesis of 13 was simply to assess whether
esters other than the ethyl ester of 7 could also be expected to
be solids.
The outcome of the study clearly demonstrates that altering
the length of the 40-polyether backbone can have a profound
effect on the ligand’s ICE, biliary ferrokinetics, and physio-
chemical properties. The results strongly suggest that the 3,6-
dioxaheptyloxypolyetherligand (6) should be pursued further
as a clinical candidate.
Synthetic Methods. (S)-4,5-Dihydro-2-[2-hydroxy-4-(3,6,9,12-
tetraoxatridecyloxy)phenyl]-4-methyl-4-thiazolecarboxylic Acid
(5). A solution of 50% (w/w) NaOH (7.0 g, 87 mmol) in CH3OH
(75 mL) was added to 11 (3.64 g, 7.72 mmol) in CH3OH (85 mL)
at 0 °C over 3 min. The reaction mixture was stirred at 0 °C for
1.5 handat roomtemperaturefor 18 h, andthe bulkofthe solvent
was removed under reduced pressure. The residue was treated
with H2O (90 mL) and was extracted with CHCl3 (4 ꢀ 50 mL).
The aqueous layer was cooled in ice, combined with saturated
NaCl (45 mL) and cold 5 N HCl (22 mL), and was extracted with
EtOAc (100 mL, 5 ꢀ 70 mL). The EtOAc layerswere washed with
saturated NaCl (75 mL). Solvent was removed in vacuo, afford-
ing 3.20 g of 5 (94%) as a yellow oil: [R] þ47.6° (c 0.86). 1H NMR
(CDCl3 þ 1-2 drops D2O) δ 1.69 (s, 3 H), 3.21 (d, 1 H, J = 11.3),
3.38 (s, 3 H), 3.53-3.57 (m, 2 H), 3.62-3.69 (m, 8 H), 3.70-3.73
(m, 2 H), 3.82-3.87 (m, 3 H), 4.11-4.15 (m, 2 H), 6.45 (dd, 1 H,
J = 8.8, 2.5), 6.50 (d, 1 H, J = 2.4), 7.27 (d, 1 H, J = 9.0). 13C
NMR δ 24.67, 39.90, 59.11, 69.66, 70.53, 70.67, 70.69, 70.71,
70.94, 72.02, 82.93, 101.56, 107.70, 109.80, 131.85, 161.32, 163.30,
171.76, 176.19. HRMS m/z calcdforC20H30NO8S, 444.1687 (M þ
H); found, 444.1691. Anal. (C20H29NO8S) C, H, N.
(S)-4,5-Dihydro-2-[2-hydroxy-4-(3,6-dioxaheptyloxy)phenyl]-
4-methyl-4-thiazolecarboxylic Acid (6). A solution of 50% (w/w)
NaOH (2.1 mL, 40 mmol) in CH3OH (20 mL) was added to 7
(1.2 g, 3.1 mmol) in CH3OH (30 mL) at 0 °C. The reaction mix-
ture was stirred at room temperature for 6 h, and the bulk of the
solvent was removed under reduced pressure. The residue was
treated with dilute NaCl (30 mL) and was extracted with ether
(2 ꢀ 20 mL). The aqueous layer was cooled in ice, acidified with
6 N HCl to pH = 2, and extracted with EtOAc (4 ꢀ 25 mL). The
EtOAc layers were washed with saturated NaCl (50 mL). Solvent
was removed in vacuo, and recrystallization from EtOAc/hexanes
furnished 0.880 g of 6 (80%) as a solid, mp 82-83 °C: [R] þ59.6°
(c 0.094). 1H NMR δ 1.70 (s, 3 H), 3.22 (d, 1 H, J = 11.2), 3.40
(s, 3 H), 3.58-3.60 (m, 2 H), 3.71-3.73 (m, 2 H), 3.83-3.87 (m þ
d, 3 H, J = 12.0), 4.15 (t, 2 H, J = 5.2), 6.45 (dd, 1 H, J = 8.8, 2.0),
6.51 (d, 1 H, J = 2.0), 7.28 (d, 1 H, J = 8.4). 13C NMR δ 24.58,
39.77, 59.13, 67.64, 69.61, 70.77, 71.99, 82.63, 101.53, 107.73,
109.63, 131.88, 161.42, 163.40, 171.96, 176.91. HRMS m/z calcd
for C16H22NO6S, 356.1162 (M þ H); found, 356.1190. Anal.
(C16H21NO6S) C, H, N.
Ethyl (S)-4,5-Dihydro-2-[2-hydroxy-4-(3,6-dioxaheptyloxy)phen-
yl]-4-methyl-4-thiazolecarboxylate (7). Flame activated
K2CO3 (2.16 g, 15.6 mmol) and 1249 (3.97 g, 14.5 mmol) were
added to 1048 (4.0 g, 14.2 mmol) in acetone (100 mL). The
reaction mixture was heated at reflux for 2 d. After cooling to
room temperature, the solids were filtered and washed with
acetone, and the filtrate was concentrated by rotary evapora-
tion. The residue was treated with 1:1 0.5 M citric acid/saturated
NaCl (100 mL) and was extracted with EtOAc (3 ꢀ 50 mL). The
organic extracts were washed with H2O (100 mL) and saturated
NaCl (100 mL). After solvent was removed in vacuo, recrystalli-
zation from EtOAc/hexanes furnished 3.97 g of 7 (73%) as a
solid, mp 68-70 °C: [R] þ47.4° (c 0.114). 1H NMR δ 1.30 (t, 3 H,
J = 7.2), 1.66 (s, 3 H), 3.19 (d, 1 H, J = 11.2), 3.40 (s, 3 H),
3.57-3.59 (m, 2 H), 3.71-3.73 (m, 2 H), 3.83-3.88 (d þ m, 3 H,
J = 11.6), 4.16 (t, 2 H, J = 4.8), 4.24 (dq, 2 H, J = 7.2, 1.6), 6.46
(dd, 1 H, J = 8.8, 2.4), 6.49 (d, 1 H, J = 2.8), 7.29 (d, 1 H, J =
8.4), 12.69 (s, 1 H). 13C NMR δ 14.12, 24.48, 39.84, 59.09, 61.89,
67.55, 69.52, 70.80, 71.94, 83.12, 101.45, 107.28, 109.89, 131.69,
161.18, 162.99, 170.81, 172.80. HRMS m/z calcd for C18H26-
NO6S, 384.1475 (M þ H); found, 384.1509. Anal. (C18H25-
NO6S) C, H, N.
Experimental Section
Materials. Reagents were purchased from Aldrich Chemical
Co. (Milwaukee, WI). Fisher Optima grade solvents were
routinely used, and DMF was distilled. Reactions were run
under a nitrogen atmosphere, and organic extracts were dried
with sodium sulfate. Silica gel 40-63 from SiliCycle, Inc.
(Quebec City, Quebec, Canada) was used for column chroma-
tography. Melting points are uncorrected. Glassware that was
presoaked in 3 N HCl for 15 min, washed with distilled water
and distilled EtOH, and oven-dried was used during the iso-
lation of 5 and 6. Optical rotations were run at 589 nm (sodium
D line) and 20 °C on a Perkin-Elmer 341 polarimeter, with c
being concentration in grams of compound per 100 mL of
CHCl3. 1H NMR spectra were run in CDCl3 at 400 MHz, and
chemical shifts (δ) are given in parts per million downfield from
tetramethylsilane. Coupling constants (J) are in hertz. 13C NMR
spectra were measured in CDCl3 at 100 MHz, and chemical
shifts (δ) are given in parts per million referenced to the residual
solvent resonance of δ 77.16. The base peaks are reported for the
ESI-FTICR mass spectra. Elemental analyses were performed
by Atlantic Microlabs (Norcross, GA) and were within (0.4%
of the calculated values. Purity of the compounds is supported
by elemental analyses and high pressure liquid chromatography
(HPLC). In every instance, the purity was g95%.
Cebus apella monkeys were obtained from World Wide
Primates (Miami, FL). Male Sprague-Dawley rats were pro-
cured from Harlan Sprague-Dawley (Indianapolis, IN). Ultra-
pure salts were obtained from Johnson Matthey Electronics
(Royston, UK). All hematological and biochemical studies41
were performed by Antech Diagnostics (Tampa, FL). Atomic
absorption (AA) measurements were made on a Perkin-Elmer
model 5100 PC (Norwalk, CT). Histopathological analysis was
carried out by Florida Vet Path (Bushnell, FL).
X-ray Experimental. Data for 6 and 7 were collected at 173 K
on a Siemens SMART PLATFORM equipped with a CCD
area detector and a graphite monochromator utilizing MoKR
radiation (λ = 0.71073 A). Cell parameters were refined using
up to 8192 reflections. A full sphere of data (1850 frames) was
collected using the ω-scan method (0.3° frame width). The first
50 frames were remeasured at the end of data collection to
monitor instrument and crystal capability (maximum correction
on I was <1%). Absorption corrections by integration were
applied based on measured indexed crystal faces.
The structures were solved by the Direct Methods in
SHELXTL657 and refined using full-matrix least-squares. The
non-H atoms were treated anisotropically, whereas the hydro-
gen atoms were calculated in ideal positions and were riding on
their respective carbon atoms. For 6, a total of 227 parameters
were refined in the final cycle of refinement using 3588 reflec-
tions with I > 2σ(I) to yield R1 and wR2 of 3.16% and 8.58%,
respectively. For compound 7, a total of 243 parameters were
refined in the final cycle of refinement using 4082 reflections
with I > 2σ(I) to yield R1 and wR2 of 2.52% and 6.53%,
respectively. Refinements were done using F2. Full crystallo-
graphic data for 6 and 7 have been submitted to CCDC
(deposition nos. CCDC 757291 and 757292).
13-Iodo-2,5,8,11-tetraoxatridecane (9). Sodium iodide (8.61 g,
57.5 mmol) was added to a solution of 8 (10.37 g, 28.61 mmol) in
acetone (230 mL), and the reaction mixture was heated at reflux
for 18 h. After the solvent was evaporated in vacuo, the residue
was combined with H2O (150 mL) and was extracted with
CH2Cl2 (150 mL, 2 ꢀ 80 mL). The organic extracts were washed
with 1% NaHSO3 (80 mL), H2O (80 mL), and saturated NaCl

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2851
(50 mL), and solvent was evaporated in vacuo. Purification by
flash column chromatography using 14% acetone/CH2Cl2 gen-
erated 8.56 g of 9 (94%) as a colorless liquid: 1H NMR δ
3.24-3.29 (m, 2 H), 3.39 (s, 3 H), 3.54-3.58 (m, 2 H),
3.64-3.70 (m, 10 H), 3.74-3.78 (m, 2 H). 13C NMR δ 59.17,
70.32, 70.65, 70.70, 70.73, 70.77, 72.05, 72.09. HRMS m/z calcd
for C9H20IO4, 319.0401 (M þ H); found, 319.0417. Anal.
(C9H19IO4) C, H.
tional 5 days after the drug was given. Iron concentrations were
determined by flame absorption spectroscopy as presented in
other publications.40,62
Drug Preparation and Administration. In the iron clear-
ing experiments, the rats were given 5-7 orally at a dose of
300 μmol/kg. Ligand 5 was given by gavage as its monosodium
salt (prepared by the addition of 1 equiv of NaOH to a suspen-
sion of the free acid in distilled water), while 6 and 7 were given in
capsules. The primates were given 5-7 orally at a dose of
75 μmol/kg. Ligand 5 was given to the primates by gavage as
its monosodium salt. Analogue 6 was given to the monkeys by
gavage as its monosodium salt, as well as in capsules. Ligand 7
was given to the monkeys in capsules. Drug preparation for the
rodent toxicity studies of 6 and 7 are described below.
Calculation of Iron Chelator Efficiency. The theoretical iron
outputs of the chelators were generated on the basis of a 2:1
ligand:iron complex. The efficiencies in the rats and monkeys
were calculated as set forth elsewhere.42,58 Data are presented as
the mean ( the standard error of the mean; p-values were
generated via a one-tailed Student’s t-test in which the inequality
of variances was assumed, and a p-value of <0.05 was con-
sidered significant.
Plasma Analytical Methods. Analytical separation was per-
formed on a Discovery RP Amide C16 HPLC system with a
Shimadzu SPD-10A UV-vis detector at 310 nm as previously
described.51,58 Mobile phase and chromatographic conditions
were as follows. Mobile phase A (MPA): 25 mM KH2PO4 þ
2.5 mM 1-octanesulfonic acid, pH 3 (95%) and acetonitrile
(5%); mobile phase B (MPB): 25 mM KH2PO4 þ 2.5 mM
1-octanesulfonic acid, pH 3 (40%) and acetonitrile (60%). The
chelator concentrations were calculated from the peak area
fitted to calibration curves by nonweighted least-squares linear
regression with Shimadzu CLASS-NP 7.4 chromatography
software. The method had a detection limit of 0.1 μM and was
reproducible and linear over a range of 0.2-20 μM.
The ethyl ester (7) was solubilized in DMSO and further
diluted with distilled water to provide a 100 μM solution. A
25 μL aliquot of the drug solution was added to centrifuge tubes
containing 100 μL of rat or primate plasma. Control experi-
ments were also performed in which saline was used in place of
the rat or monkey plasma. The centrifuge tubes were vortexed
and incubated in a shaking incubator at 37 °C for 1 or 2 h. Note
that separate samples were processed for each species at each
time point (four samples total). Methanol (400 μL) was added to
the centrifuge tubes at the end of the incubation period to stop
the reaction. The tubes were stored at -20 °C for at least 0.5 h.
The tubes were then allowed to warm to room temperature. The
samples were vortexed and centrifuged for 10 min at 10000 rpm.
Supernatant (100 μL) was diluted with MPA (minus the
1-octanesulfonic acid, 400 μL), vortexed, and run on the HPLC
as usual.
Ethyl (S)-4,5-Dihydro-2-[2-hydroxy-4-(3,6,9,12-tetraoxatri-
decyloxy)phenyl]-4-methyl-4-thiazolecarboxylate (11). Flame ac-
tivated K2CO3 (0.666 g, 4.82 mmol) was added to a solution of 9
(1.46 g, 4.59 mmol) and 1048 (1.08 g, 3.84 mmol) in acetone
(85 mL), and the reaction mixture was heated at reflux for 43 h.
After cooling to room temperature, the solids were filtered and
washed with acetone, and the filtrate was concentrated by rotary
evaporation. The residue was combined with 1:1 0.5 M citric
acid/saturated NaCl (100 mL) and was extracted with EtOAc
(3 ꢀ 80 mL). The organic extracts were washed with 1%
NaHSO3 (80 mL), H2O (80 mL) and saturated NaCl (55 mL).
After solvent was removed in vacuo, the residue was purified
by flash column chromatography eluting with 25% acetone/
petroleum ether then 9% acetone/CH2Cl2, furnishing 1.33 g of 11
(73%) as a yellow oil: [R] þ36.2° (c 1.20). 1H NMR δ 1.30 (t, 3 H,
J = 7.2), 1.66 (s, 3 H), 3.19 (d, 1 H, J = 11.3), 3.38 (s, 3 H),
3.52-3.56 (m, 2 H), 3.62-3.74 (m, 10 H), 3.81-3.88 (m, 3 H),
4.12-4.16 (m, 2 H), 4.20-4.28 (m, 2 H), 6.46 (dd, 1 H, J = 8.6,
2.3), 6.49 (d, 1 H, J = 2.4), 7.29 (d, 1 H, J = 8.6). 13C NMR δ
14.21, 24.59, 39.95, 59.14, 62.01, 67.66, 69.58, 70.62,
70.71, 70.73, 70.97, 72.04, 83.23, 101.52, 107.42, 109.99,
131.78, 161.28, 163.11, 170.90, 172.95. HRMS m/z calcd for
C22H34NO8S, 472.2000 (M þ H); found, 472.2007. Anal.
(C22H33NO8S) C, H, N.
Isopropyl (S)-4,5-Dihydro-2-[2-hydroxy-4-(3,6-dioxaheptyloxy)-
phenyl]-4-methyl-4-thiazolecarboxylate (13). 2-Iodopropane
(1.60 g, 9.41 mmol) and DIEA (1.22 g, 9.44 mmol) were
successively added to 6 (2.1 g, 5.9 mmol) in DMF (50 mL),
and the reaction mixture was stirred at room temperature for
72 h. After solvent removal under high vacuum, the residue was
treated with 1:1 0.5 M citric acid/saturated NaCl (100 mL) and
was extracted with EtOAc (3 ꢀ 100 mL). The organic extracts
were washed with 50 mL portions of 1% NaHSO3, H2O, and
saturated NaCl, and solvent was evaporated in vacuo. Purifica-
tion by flash column chromatography using 5% acetone/CH2Cl2
generated 1.99 g of 13 (85%) as a yellow oil: [R] þ40.0° (c 0.125).
1H NMR δ 1.26 and 1.27 (2 d, 6 H, J = 5.5), 1.63 (s, 3 H), 3.17 (d,
1 H, J = 11.2), 3.38 (s, 3 H), 3.55-3.58 (m, 2 H), 3.69-3.72 (m, 2
H), 3.81-3.86 (d þ m, 3 H, J = 11.2), 4.15 (t, 2 H, J = 5.2), 5.07
(septet, 1 H, J = 6.4), 6.46(dd, 1 H, J = 9.2, 2.0), 6.49 (d, 1 H, J =
2.4), 7.28 (d, 1 H, J = 8.4), 12.7 (br s, 1 H). 13C NMR δ 21.54,
24.27, 39.63, 58.98, 67.46, 69.35, 69.42, 70.69, 71.85, 83.10,
101.37, 107.14, 109.83, 131.57, 161.11, 162.88, 170.55, 172.10.
HRMS m/z calcd for C19H28NO6S, 398.1637 (M þ H); found,
398.1658. Anal. (C19H27NO6S) C, H, N.
Toxicity Evaluation of 6 and 7 in Rodents. Male Sprague-
Dawley rats (300-350 g) were fasted overnight and were given
the chelators first thing in the morning. The rats were fed ∼3 h
postdrug and had access to food for ∼5 h before being fasted
overnight. Ligand 6 was given to the rats orally once daily
at a dose of 384 μmol/kg/d ꢀ 10 d. Note that this dose is
equivalent to 100 mg/kg/d of the DFT sodium salt. The chelator
(6) was administered orally in gelatin capsules (n = 5), or by
gavage as its monosodium salt (n = 10). The ethyl ester (7) was
administered orally in capsules once daily at a dose of
192 (n=6) or 384 μmol/kg/d (n=5) ꢀ 10 d. Age-matched rats
(n = 12) served as untreated controls. The rats were eutha-
nized 24 h postdrug (day 11) and extensive tissues were collec-
ted for histopathological analysis. Samples of the kidney, liver,
heart, and pancreas were reserved and assessed for their iron
content.
Biological Methods. Cannulation of Bile Duct in Non Iron-
Overloaded Rats. The cannulation has been described pre-
viously.40,41,58 Bile samples were collected from male Sprague-
Dawley rats (400-450 g) at 3 h intervals for up to 48 h. The urine
sample(s) was taken at 24 h intervals. Sample collection and
handling are as previously described.40,41
Iron Loading of C. apella Monkeys. The monkeys (3.5-4 kg)
were iron overloaded with intravenous iron dextran as specified
in earlier publications to provide about 500 mg of iron per kg of
body weight;40,59 the serum transferrin iron saturation rose to
between 70 and 80%. At least 20 half-lives, 60 days,60 elapsed
before any of the animals were used in experiments evaluating
iron-chelating agents.
Primate Fecal and Urine Samples. Fecal and urine samples
were collected at 24 h intervals and processed as described
previously.40,41,61 Briefly, the collections began 4 days prior to
the administration of the test drug and continued for an addi-
Preparation of Rodent Tissues for the Determination of their
Iron Content. The initial step in the tissue preparation involved
removing any obvious membranes or fat. A sample of each

2852 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Bergeron et al.
tissue (300-350 mg) was weighed and transferred to acid-
washed hydrolysis (pressure) tubes. Note that the same region
of each tissue was always utilized. Concentrated HNO3 (65%),
1.5 mL, and distilled water (2 mL) were added. The tubes were
then sealed and placed in a 120 °C oil bath for 5 h; the tubes were
vented as necessary. Then, the tubes were removed from the oil
bath and allowed to cool to room temperature. The temperature
of the oil bath was decreased to 100 °C. Once the samples were
cooled, 0.7 mL of hydrogen peroxide (30%) was added to the
hydrolysis tube. The samples were placed back in the oil bath
and cooked overnight. The samples were then removed from the
oil bath and allowed to cool to room temperature. The hydroly-
sis tubes were vortexed, and the digested samples were poured
into 50 mL volumetric flasks. The samples were brought to
volume using distilled water. Finally, the samples were poured
into a syringe and filtered using 0.45 μ, 30 mm, Teflon syringe
filters. Iron concentrations were determined by flame absorp-
tion spectroscopy as presented in other publications.40,41
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2008; http://www.pharma.us.novartis.com/product/pi/pdf/desferal.pdf.
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Acknowledgment. The project described was supported by
grant number R37DK049108 from the National Institute of
Diabetes and Digestive and Kidney Diseases. The content is
solely the responsibility of the authors and does not necessar-
ily represent the official views of the National Institute of
Diabetes and Digestive and Kidney Diseases or the National
Institutes of Health. We thank Elizabeth M. Nelson and Katie
Ratliff-Thompson for their technical assistance and Miranda
E. Coger for her editorial and organizational support. K.A.A.
acknowledges the National Science Foundation and the Uni-
versity of Florida for funding of the purchase of the X-ray
equipment. We acknowledge the spectroscopy services in the
Chemistry Department, University of Florida, for the mass
spectrometry analyses.
Supporting Information Available: Elemental analytical data
for synthesized compounds, X-ray crystallographic data collec-
tion and refinement parameters along with selected bond angles
and lengths for compounds 6 and 7. This material is available
free of charge via the Internet at http://pubs.acs.org.
(23) Olivieri, N. F. Long-Term Therapy with Deferiprone. Acta Hae-
matol. 1996, 95, 37–48.
(24) Olivieri, N. F.; Brittenham, G. M.; McLaren, C. E.; Templeton, D.
M.; Cameron, R. G.; McClelland, R. A.; Burt, A. D.; Fleming, K.
A. Long-Term Safety and Effectiveness of Iron-Chelation Therapy
with Deferiprone for Thalassemia Major. N. Engl. J. Med. 1998,
339, 417–423.
(25) Richardson, D. R. The Controversial Role of Deferiprone in
the Treatment of Thalassemia. J. Lab. Clin. Med. 2001, 137, 324–
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